Phenobarbital Induces Cell Cycle Transcriptional Responses in Mouse Liver Humanized for Constitutive Androstane and Pregnane X Receptors

Abstract

The constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) are closely related nuclear receptors involved in drug metabolism and play important roles in the mechanism of phenobarbital (PB)-induced rodent nongenotoxic hepatocarcinogenesis. Here, we have used a humanized CAR/PXR mouse model to examine potential species differences in receptor-dependent mechanisms underlying liver tissue molecular responses to PB. Early and late transcriptomic responses to sustained PB exposure were investigated in liver tissue from double knock-out CAR and PXR (CAR(KO)-PXR(KO)), double humanized CAR and PXR (CAR(h)-PXR(h)), and wild-type C57BL/6 mice. Wild-type and CAR(h)-PXR(h) mouse livers exhibited temporally and quantitatively similar transcriptional responses during 91 days of PB exposure including the sustained induction of the xenobiotic response gene Cyp2b10, the Wnt signaling inhibitor Wisp1, and noncoding RNA biomarkers from the Dlk1-Dio3 locus. Transient induction of DNA replication (Hells, Mcm6, and Esco2) and mitotic genes (Ccnb2, Cdc20, and Cdk1) and the proliferation-related nuclear antigen Mki67 were observed with peak expression occurring between 1 and 7 days PB exposure. All these transcriptional responses were absent in CAR(KO)-PXR(KO) mouse livers and largely reversible in wild-type and CAR(h)-PXR(h) mouse livers following 91 days of PB exposure and a subsequent 4-week recovery period. Furthermore, PB-mediated upregulation of the noncoding RNA Meg3, which has recently been associated with cellular pluripotency, exhibited a similar dose response and perivenous hepatocyte-specific localization in both wild-type and CAR(h)-PXR(h) mice. Thus, mouse livers coexpressing human CAR and PXR support both the xenobiotic metabolizing and the proliferative transcriptional responses following exposure to PB.

Small-molecule modulators of the constitutive androstane receptor

“…Studies have also been conducted in transgenic humanized knockout models (mice in which mCAR was replaced with hCAR), with interesting results. Although some researchers have reported that PB can produce proliferative transcriptional responses [113] and promote some tumor production [114] in hCAR/hPXR double-humanized mice, others have demonstrated liver hypertrophy without hyperplasia [115] and conclude that humanized mouse models are relatively resistant to tumorigenesis [92, 110, 114, 116, 117]. However, the hCAR/hPXR transgenic mouse, in which human-origin CAR and PXR are used with mouse-origin CAR target genes, may not be an appropriate model for extrapolating risk from rodent findings to humans.…”

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“…For the CAR and PXR knockout mice, no pathway deregulation is observed as expected due to the lack of deregulation of individual genes. These results obtained with MARCARviz are in concordance with the results of the analysis performed by Luisier et al (2014). To visualize the major effects of phenobarbital on gene expression in wild type, humanized CAR/PXR, and CAR/PXR knockout mice, we used the "Heat maps" tool, using the same filtering (two-fold deregulation and corrected limma p-value  0.05) and clustering of both genes and conditions (see Fig.…”

“…The animal experiments have been approved by the respective ethics committees and were performed according to established experimental guidelines. Study design and raw data generation have been previously described (Unterberger et al, 2014;Riegler et al, 2015;Braeuning et al, 2010;Luisier et al, 2014;Eichner et al, 2014;Lempiäinen et al, 2011;Ellinger-Ziegelbauer et al, 2008;Braeuning et al, 2016). These datasets cover three species: Mus musculus and Rattus norvegicus (in vivo), and Homo sapiens (in vitro).…”

“…The dataset is available from the Gene Expression Omnibus under the accession number GSE60684. An extensive analysis of this dataset was previously published by Luisier et al (2014). This use case is also available as an interactive example online at the MARCARviz web platform.…”

Pregnane X Receptor and Cancer: Context-Specificity is Key

“…PXR is important for liver regeneration (Dai et al, 2008). PXR activation induces hepatic proliferation and/or inhibits apoptosis through several mechanisms (Elcombe et al, 2012a; Elcombe et al, 2012b; Elcombe et al, 2010; Luisier et al, 2014; Xie et al, 2000). However, PXR activation also induces differentiation of osteoblasts and apoptosis of osteoclasts and certain leukemia cells (Austin et al, 2015; Hassen et al, 2014; Igarashi et al, 2007; Kameda et al, 1996; Tabb et al, 2003), suggesting that control of cell proliferation by PXR is likely tissue and cell-specific.…”